Fluorescent vasoactive intestinal peptide (VIP)

ABSTRACT

Light emitting compounds of the formula:                    
     wherein R 1  is a light-emitting moiety and R 2  is a VIP-based peptide, fragment, derivative or analog thereof, said peptide being linked at an amino acid position to (C—X), wherein (C—X) is selected from the group consisting of C═O, C═S, CH(OH), C═C═O, C═NH, CH 2 , CH(OR), CH(NR), CH(R), CR 3 R 4 , and C(OR 3 )OR 4  where R, R 3 , and R 4  are alkyl moieties or substituted alkyl moieties, wherein said compound exhibits substantial biological activity in the presence of a receptor having affinity for VIP-based peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Ser. No.08/682,810 filed Jul. 10, 1996 and entitled “Fluorescent Peptides”, U.S.Pat. No. 6,528,434 which is a continuation-in-part application of U.S.Ser. No. 08/504,856, having the same name and filed Jul. 20, 1995 nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates to peptide-based compounds having light-emittingmoieties. Peptides may be chemically linked with detectable “labels” andused, for example, to monitor peptide, cytokine, drug, and hormonereceptors at the cellular level. Typically, the labeled peptide isplaced in contact with a tissue or cell culture where it binds to anavailable receptor. Once bound, the label is detected, allowingproperties such as receptor distribution or receptor binding kinetics tobe monitored.

Peptides are typically labeled with radioactive elements such as ¹²⁵I or³H. In this case, emission of high-energy radioactive particles ismonitored using standard (γ-ray detectors, thereby allowing detection ofthe label. While detection techniques for ¹²⁵I and ³H are well-known,radioactive compounds by nature have limited half lives, and are oftenboth toxic and expensive. Moreover, current detection technology makesit difficult or impossible to detect radioactive probes in real-time,thereby precluding study of kinetic processes.

Vasoactive intestinal peptide (VIP) is a 28 amino acid peptide, firstisolated in the lung and intestine, but also found in tissues of thebrain, liver, pancreas, smooth muscle and lymphocytes. VIP is aparticularly desirable peptide to label and use to monitor cellreceptors, as this compound exhibits multiple biological roles includingvasodilation, electrolyte secretion, modulation of immune function andneurotransmission. In particular, VIP is involved in the regulation ofthe cardiovascular system by exerting vasodilation, hypotension,positive chronotropic and ionotropic effects.

SUMMARY OF THE INVENTION

The present invention provides a compound containing a VIP peptide and alight-emitting moiety that is both biologically active and opticallydetectable. The peptide is chemically attached to the light-emittingmoiety at an amino acid position that is not involved in binding to thepeptide receptor. In this way, the peptide's affinity for the bindingsite is not significantly decreased, and the compound retains highbiological activity and can be easily detected using standard opticalmeans.

In general, in one aspect, the invention provides a biologically activecompound of the formula:

where R₁ is a light-emitting moiety and R₂ is a VIP-based peptide andfragment, derivative or analog thereof. The peptide is linked at anamino acid position to (C—X) which, in turn, is selected from the groupincluding C═O, C═S, CH(OH), C═C═O, C═NH, CH₂, CH(OR), CH(NR), CH(R),CR₃R₄, and C(OR₃)OR₄ where R, R₃, and R₄ are alkyl moieties orsubstituted alkyl moieties. Preferably, the compound exhibitssubstantial biological activity in the presence of receptors havingaffinities for VIP-based peptides. The compound may also be in the formof a pharmaceutically acceptable salt or complex thereof. Preferably,the fifteenth amino acid position which is the lysine 15 amino acidresidue is bound to (C—X) is included of the VIP-based peptide.

In other preferred embodiments, the VIP-based peptide includes the aminoacid sequenceHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn.(SEQ. ID NO: 1) and the Lys 15 residue is attached to the (C—X) moiety.The lysine residue preferably is chemically bound to the (C—X) moietythrough the ε amine group of the lysine residue. In still otherpreferred embodiments, the (C—X) bond is either C═O or C═S. In otherpreferred embodiments, the peptide may be amidated at the C-terminus.

In other preferred embodiments, the light-emitting moiety (R₁) isselected from the group including4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, fluorescein, FTC, Texas red,phycoerythrin, rhodamine, carboxytetramethylrhodamine,4′6-diamidino-2-phenylindole (DAPI), indopyras dyes, Cascade blue,coumarins, nitrobenzofurazane (NBD), Lucifer Yellow, propidium iodide,CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates, lanthanidechelates, non-fluorescent dialdehydes (OPA, NDA, ADA, ATTOTAG reagentsfrom Molecular Probes) which react with primary amines (N-term lysine)in the presence of a nucleophile (i.e. CN) to form fluorescentisoindoles, dansyl dyes fluorescamine and dabcyl chloride,5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, longlifetime dyes comprised of metal-ligand complexes (MLC) which consist ofa metal center (Ru, Re, Os) and organic or inorganic ligands complexedto the metal such as [Ru(bpy)₃]²⁺ and [Ru(bpy)₂(dcbpy)], and the likeand derivatives thereof. The light-emitting moiety can be attached tothe peptide by reaction of a reactive side group (of the light-emittingmoiety) with the N-terminus amino acid of VIP. Suitable linking moietiesinclude, by way of example only, indoacetamide, maleimide,isothiocyanate, succinimidyl ester, sulfonyl halide, aldehydes, glyoxal,hydrazine and derivatives thereof.

By “VIP-based compound” is meant a peptide which includes VIP, fragmentsof VIP, derivatives or analogs thereof. VIP-based peptides may bepeptides whose sequences differ from VIP's wild-type sequence by onlyconservative amino acid substitutions. For example, one amino acid maybe substituted for another with similar characteristics (e.g., valinefor glycine, arginine for lysine, etc.) or by one or morenon-conservative amino acid substitutions, deletions, or insertionswhich do not abolish the peptide's biological activity. Other usefulmodifications include those which increase VIP's stability. The peptidemay contain, for example, one or more non-peptide bonds (which replace acorresponding peptide bond) or D-amino acids in the peptide sequence.Additionally, the C-terminus carboxylic acid group may be modified toincrease peptide stability. For example, as described above, theC-terminus may be amidated or otherwise derivatized to reduce thepeptide susceptibility to degradation.

In all cases, by “substantially biologically active” is meant thecompound binds to a receptor having an affinity IC50 value for thecompound which is no more than 15 times, more preferably no more than 10times and most preferably equal to or less than that of thecorresponding unlabeled peptide. Receptor affinity in this case can bedetermined using known methods, such as methods involving competitivebinding of radioactively labeled peptides or by using known methods offluorescence polarization or other known fluorescence technique formeasuring the K_(d) for the receptor/peptide interaction.

By “low” or “no” biological activity or “biologically inactive” is meantbiological activities less than 1.0% of the biological activity of R₂—Hin the presence of a receptor having affinity for VIP.

The above-identified compound is useful in the labeling of cell receptorsites, cell sorting, flow cytometry and performing fluoroimmunoassays.In another aspect, the invention provides a method for labeling areceptor having an affinity for a VIP-based peptide by contacting thereceptor with one or more of the compounds described above. Cellreceptor sites, can be imaged by contacting candidate cell receptorsites with the compound of the invention, and then detecting the boundcompounds as an indication of the cell receptor sites. Cell sorting canbe performed by contacting a population of cells with compound andisolating cells bound to the compound. Flow cytometry can be performedby contacting a population of cells with the compound and detectingcells bearing receptors on their surfaces by detecting cells bound tothe compound.

The invention has many advantages. In a general sense,peptide-containing compounds which retain their biological activityafter being labeled with light-emitting moieties have a wide variety ofbiological applications. Such compounds can be used to identify,visualize, quantify, target and select receptors on cells and tissuesboth in vitro and in vivo. These compounds may be used in place of moreconventional labeled peptides, such a ¹²⁵I radiolabeled peptides.Radiolabeled compounds are often toxic, environmentally hazardous,chemically unstable and have, by the nature of the radioactive decayrate, relatively short lifetimes. In contrast, fluorescently-labeled VIPis relatively safe and non-toxic, thereby allowing it to be synthesizedand used without employing special laboratory procedures. Similarly,following use, fluorescent VIP may be easily disposed, whereas disposalof radioactive compounds is both time-consuming and costly. In addition,fluorescent markers for VIP receptors are stable and may be stored forextensive periods of time without undergoing extensive degradation.

Use of VIP in the labeled compound is also advantageous. As describedabove, VIP exhibits biological activity in various and is involved inregulation of the cardiovascular system. In addition, this peptide has arelatively simple structure (28 amino acids) and can be synthesized andisolated with standard, well-known techniques.

During typical experiments, fluorescent markers for VIP receptors emitoptical signals, and thus may be monitored by eye or with the aid ofexternal optical detectors. In this way, the fluorescent peptidesobviate the need for secondary detection steps sometimes used forradiolabeled compounds or incubation with secondary labeled compounds.Detection of optical radiation is, in general, relatively simple andcost-effective compared to detection of radioactive particles (e.g.,γ-particles); conventional charge-coupled device (CCDs) orlight-sensitive cameras can therefore be used without modification forthis application.

In addition, because of their high optical emission rates andwell-characterized emission cross sections, fluorescent markers attachedto VIP receptors can be used for real-time, quantified imaging of anumber of dynamic biological phenomena, such as kinetics associated withreceptor binding. The compounds can also be used for static processes,such as monitoring peptide distribution within a cell. VIP receptorsmarked with fluorescent peptides may also be used in flow cytometry,confocal microscopy, fluorescence polarization spectroscopy, and anyother techniques exploiting the optical detection of fluorescence orphotoluminescence.

Other advantages and features of the invention will become apparent fromthe following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the chemical structure offluorescently-labeled peptide according to the invention; and

FIG. 2 is an IC50 curve showing displacement of ¹²⁵I labeled VIP byincreasing concentration of unlabeled and fluorescently labeled VIP.

DETAILED DESCRIPTION OF THE INVENTION

Referring first to FIG. 1, in a preferred embodiment, a fluorescentpeptide 10 according to the invention includes a light-emitting moiety12, such as a fluorescent dye, linked via a (C—X) bond to a peptidemoiety 14. The peptide moiety 14 includes amino acid residues of the VIPpeptide, and fragments, derivatives or analogs thereof. Region 18 ispresumed to be involved in the biological activity of the peptidecomplex.

In order to retain substantial biological activity and affinity for VIPreceptors, the peptide is attached to the (C—X) linking moiety at thefirst amino acid residue which is in a region 16 which is notbiologically active, i.e., it is not significantly involved in thepeptide's biological role. In this manner, the light-emitting moietydoes not sterically hinder or otherwise significantly affect the regioninvolved in receptor binding and the biological activity of the compoundis thus maintained. For example, in a preferred embodiment, the peptidemoiety is attached via an ε amine group of the Lys15 residue to thelight emitting moiety through a (C═O) bond. The light-emitting moietymay be covalently bonded to the (C—X) linking group at any availableposition. The (C—X) linking group represents the bond formed between thelight-emitting moiety and the peptide upon reaction and this bond mayinclude groups such as C═O, C═S, CH(OH), C═C=O, C═NH, CH₂, CH(OR),CH(NR), CH(R), CR₃R₄, and C(OR₃)OR₄ where R, R₃, and R₄ are alkylmoieties or substituted alkyl moieties.

Fluorescent peptides of this type have amino acids which are availablefor binding to VIP-recognizing receptors, thereby enabling the peptideto be used for labeling purposes. Once bound to an available receptor,the attached light-emitting moiety retains optical properties similar tothose of the unbound light-emitting molecule. In this way, thefluorescent peptide can bind to the corresponding receptor and emitlight following absorption of an incident optical field, and thus serveas a marker for the VIP receptor. This allows the receptor to be“labeled” and permits investigation, for example, of peptide/receptorinteractions. In particular, labeled peptides participating inreceptor/peptide interactions can be monitored to determine the locationof receptors in cell or tissue samples, and additionally allowquantification of receptors, determination of the receptor affinity forvarious ligands, or the identification of various populations of cells.

VIP-based peptides may be synthesized using techniques known in the art,extracted from natural systems, or obtained from commercial sources(e.g., American Peptide, Peninsula, Neosystems, Sigma, BASF). A list ofVIP analogs which may be used in practice of the invention are availablefrom American Peptide Co. product catalogue. Typically, the peptide iseither purchased or synthesized using conventional solid-phase synthetictechniques. Preferably, the peptide is substantially pure, meaning thatit is at least 60% by weight free from the other compounds with which itis naturally associated.

In general, any dye, porphyrin, fluorophore, or other light-emittingcompound may be complexed with the VIP-based peptide. In preferredembodiments, the light-emitting moiety is selected from the groupincluding 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, fluorescein, FTC,Texas red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, DAPI,indopyras dyes, Cascade blue, coumarins, NBD, Lucifer Yellow, propidiumiodide, CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates,lanthanide chelates, non-fluorescent dialdehydes (OPA, NDA, ADA, ATTOTAGreagents from Molecular Probes) which react with primary amines (N-termlys) in the presence of a nucleophile (i.e. CN) to form fluorescentisoindoles, dansyl dyes fluorescamine and dabcyl chloride,5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid, longlifetime dyes comprised of metal-ligand complexes (MLC) which consist ofa metal center (Ru, Re, Os) and organic or inorganic ligands complexedto the metal such as [Ru(bpy)₃]²⁺ and [Ru(bpy)₂(dcbpy)], and the likeand derivatives thereof. The synthesis and structures of several dyeswhich may be used are described in U.S. Pat. Nos. 5,248,782; 5,274,113;and 5,187,288, the contents of which are incorporated herein byreference. Other light-emitting moieties used in labeling or otherapplications may be attached to the peptide. For example, suitablelight-emitting moieties are described in “Handbook of Fluorescent Probesand Research Chemicals-5^(th) Edition” by Richard P. Haugland, 1994; and“Design and Application of Indicator Dyes”, Noninvasive Techniques inCell Biology: 1-20 by Richard P Haugland et al., Wiley-Liss Inc. (1990),the contents of each of which is incorporated herein by reference.

Once the desired peptide is obtained, fluorescent peptides having highbiological activities are made by attaching the light-emitting moiety tothe first amino acid position of the VIP-based moiety. In general, thisreaction is carried out by modifying a functional group on the peptide,most typically a thiol or amine group, so that this moiety may be easilyattached to the light-emitting compound. Reactions for suchmodifications are described in, for example, “Handbook of FluorescentProbes and Research Chemicals-5th Edition” (supra). In general, thiolsreact with alkylating groups (R′—Z) to yield relatively stable thiolethers (R′—S—R), e.g., (C—X) is the α-carbon of R′, with the leavinggroup Z preferably being a halogen (e.g., Cl, Br, I) and the like. Inparticular, the most common reagents for derivatization of thiols arehaloacetyl derivatives. Reaction of these reagents with thiols proceedsrapidly at or below room temperature in the physiological pH range.

The conditions used to modify amine moieties of the desired peptide willdepend on the class of amine (e.g., aromatic, aliphatic) and itsbasicity. Aliphatic amines, such as the α-amino acid groups of lysineand arginine or the amine of lysine, are moderately basic and reactivewith acylating reagents. The concentrations of the free base form of thealiphatic amines below pH 8 is very low; thus, the kinetics of acylationreaction of amines by isothiocyanates, succinimidyl esters, and otherreagents is strongly pH-dependent. Although amine acylation reactionsshould usually be carried out above pH 8.5, the acylation reagentsdegrade in the presence of water, with the rate increasing as the pHincreases. Therefore, a pH of 8.5-9.5 is usually optimal.

In general, reactive groups on the light-emitting moiety, such asunsaturated alkyl groups or acylating moieties, will react with themodified peptide to form a dye/peptide bond. The chemical structure ofthe light-emitting moiety may affect the synthetic route used tosynthesize the fluorescent VIP analog. It may be necessary, for example,to modify the light-emitting moiety so that it includes a reactive groupprior to exposing this moiety to the desired peptide. Such side groupsmay include indoacetamide, maleimide, isothiocyanate, succinimidylester, sulfonyl halide, aldehyde, glyoxal and hydrazine derivatives.Amino acids including alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine may be labeled according to the methoddescribed herein.

The chemistry used to synthesize the fluorescent peptide is not greatlydependent upon the exact structure of the VIP analog. Thus, the generalprocedure outline below may be used for most VIP-based peptides.Attachment of this peptide to a light-emitting moiety is described indetail in the Examples provided below.

Once synthesized, the resulting complex is purified, preferably using acolumn chromatography method such as high pressure liquid chromatography(HPLC). Collected fractions are then screened using analytical methodsto determine if adequate biological activity is present. Fluorescent VIPanalogs having adequate biological activities are selected by firstexposing these analogs to VIP receptors; compounds binding effectivelyto these sites are then isolated from relatively inactive fluorescentpeptides. In general, this selection process can be performed usingstandard techniques, such a column chromatography or other analyticaltechniques known in the art. The selection process is designed to allowmaintenance of the compound's pharmacological binding, and thus allowsonly the dye/peptide compounds exhibiting substantial biologicalactivities to be separated from relatively inactive compounds.

Referring now to FIG. 2, a fluoresceinyl-VIP with the amino acidsequenceHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH₂was tested to determine its affinity for VIP receptor sites. Thefluoresceinyl-VIP compound absorbs light in the range of 494 nm andfeatures an emission spectrum centered around 518 nm. The compoundexhibits comparable, dose-dependent binding to VIP receptors whencompared to native VIP peptides, as determined by displacement ofradiolabeled VIP bound to rat forebrain membranes. The concentrations atwhich 50% of the binding is inhibited (i.e., the IC50) is 2.4 nM for thefluorescein-labeled compound, as compared to 0.74 nM for the unlabeledVIP. The IC₅₀ is related to the binding constant K_(i) by the formulaIC₅₀=K_(i)(l+F_(L)/K_(d)), where F_(L) is the concentration of the freelabeled ligand and K_(d) is the dissociation constant for the labeledligand. The K_(i) for this compound was 2.3 nM for the labeled compoundversus 0.71 nM for the unlabeled compound. This demonstrates the highdegree of retention of biological activity.

In cases where it is not known which amino acid site may be complexed toform the fluorescent peptide of the invention, the peptide-fluorophorecomplexes may be screened to identify suitable complexes. The generalsynthetic method for identifying light-emitting biologically activecompounds of the invention is as follows.

The VIP-based peptide and the fluorophore of choice are incubated toform a mixture of compounds. Incubation is performed under conditionswhich permit optimal peptide labeling. Typically, a solution containingthe peptide at a concentration of about 10⁻²-10⁻⁴ M is mixed with thelight-emitting moiety in a highly basic solution, i.e., pH 9.3-10.7 suchas a carbonate buffer, in at least a 1:4 peptide:light-emitting moietymolar ratio. The solution is mixed at room temperature for a time ofbetween about 24-48 hours, and is protected from light and shakenperiodically. The resulting mixture includes biologically active andinactive whole peptides, cleaved fragments of peptides and singly andmultiply labeled peptides.

After covalent bonding of the light-emitting moiety and the peptideoccurs, unbound fluorophore is removed. In general, this step isperformed using standard techniques such as column chromatography orother analytical techniques known in the art. In a typical example,unreacted amounts of the free fluorophore are removed using a G-50column equilibrated with phosphate buffered saline (pH 7.4) and spun a3000 rpm for a period of between 0.5 and 20 minutes. The resultanteluent contains a mixture of labeled biologically active and inactivepeptides.

This solution is then collected and subjected to a high-stringencypharmacological binding assay. In this assay, only biologically activecompounds are bound to tissue receptors; inactive compounds are washedaway. The assay is typically performed on tissue sections,receptor-coated columns or membrane homogenates. In a typical example,an aliquot of the fluorescent peptide mixture is first dissolved in anaqueous solution (1:100) and incubated with an immobilized tissue samplecontaining high numbers of the peptide receptor, e.g., receptortransfected membrane homogenates.

The selection process is designed to separate compounds exhibitingsubstantial biological activity from those relatively inactivecompounds. If necessary, during the assay, binding of the biologicallyactive compounds may be rapidly observed visually, in a fluorometer orby using more sensitive techniques such as fluorescence polarizationspectroscopy.

The receptor-bound compounds are then removed from the tissue surfaceand analyzed to identify the site at which the fluorophore is attached,i.e., the site allowing fluorophore attachment without interference withreceptor binding. Biologically active compounds bound to membranereceptors are separated from the remaining inactive fluorescent peptidesin solution, either by centrifugation of membrane homogenates (typicallyat 3000 rpm for about 5 minutes) or, in the case of tissue sections, byrapidly rinsing the sections in incubation buffer at 4° C. The membranesare then resuspended in binding buffer with the biologically activecompound removed from the cell surfaces by incubation in a highsalt/acid wash solution.

Once isolated, biologically active compounds are analyzed using knowntechniques, such as carboxypeptidase digestion and capillaryelectrophoresis, laser induced capillary zone electrophoresis, massspectroscopy, and/or HPLC or amino acid sequencing, to identify the siteof attachment between the light-emitting moiety and the peptide.

Fluorescent VIP compounds selected to have high biological activity havea number of uses. For most applications, the fluorescent compound isfirst contacted with the sample of interest. The compound is thenincubated with the cell or tissues of the sample for a select timeperiod and allowed to interact with the VIP receptor. If necessary,excess, non-specifically bound compound may be washed away.

Once the peptide is bound to the desired receptor site, the labeledsample is imaged using standard techniques known in the art.Conventional microscopy methods, such as fluorescence or confocalmicroscopy, may be used to optically excite and then detect emissionfrom the labeled receptors. Other imaging techniques which can be usedwith the fluorescent VIP-based compounds include atomic forcemicroscopy, fluorescence polarization spectroscopy, and fluorimetry.

Using the above techniques, small-scale features in the cell whichnormally would be difficult or impossible to detect are observed. Forexample, this allows visualization of intracellular receptor sites.Moreover, labeled peptides participating in peptide-receptorinteractions can be monitored to determine the location of receptors, todetermine receptor affinity for various unknown ligands (drugscreening), and to identify various populations of cells endowed withpeptide receptors. Optical radiation emitted from the fluorescing moietycan be easily and rapidly detected, allowing the fluorescent peptides tobe used to monitor real-time receptor/peptide interactions. In this way,the compounds permit study of kinetic processes in the cell. Otherapplications include receptor sorting using FACS(fluorescence-associated cell sorting) and measurement of serum peptidelevels using FIA (fluorescent immunoassays) either in vivo or in vitrofor research or diagnostic purposes. In general, techniques which mayutilize the compounds of the invention include, without limitation, flowcytometry, cell sorting (for example, top isolate populations of cellsbearing a receptor of interest), tumor marking, competitive bindingassays for drug screening, fluorescent immunoassays, and other in vitroexperimental techniques involving compound labeling according totechniques known in the art.

The following Examples are used to more particularly point out thesynthesis, selection methods, and use of fluorescent VIP analogs havinghigh biological activities.

EXAMPLE 1

Synthesis of VIP peptide. Peptides were synthesized using solid phasepeptide synthesis methods either manually or automated (MPS396 peptidessynthesizer, Advanced ChemTech). Coupling of amino acid residues wasaccomplished via Fmoc peptide synthesis chemistry (Fields, et al., 1990,IJPPR 35, 161). Syntheses were performed on Wang or on amide Rinkresins, with fully side chain protected of amino acids. BOP, PyBOP orTBTU were used as activation agents depending on the chemistry anddifficulty of the coupling reaction. All chemicals were purchased fromAdvanced ChemTech, Bachem and Calbiochem/NovaBiochem. Formation of eachpeptide bond between residues of the sequence was ensured by using a 3to 6 fold excess of coupling reagents and by “double coupling”; meaningthat the coupling reaction was repeated for each amino acid added to thegrowing peptide chain.

Fluorescein Labeling of Peptides. VIP peptide was synthesized using anorthogonal protection scheme making it possible to deprotect the sidechain amino acid group of lysine 15, while leaving remaining reactivegroups in the peptide protected and the peptide attached to the resin.This deprotected lysine 15 was then labeled by mixing the resin with asolution of dye in dimethyl formamide (DMF) or NMP.

A 3-fold excess (estimated peptide in nmoles to fluorescein) of thecarboxylic form of fluorescein was used and the reaction was allowed toproceed for 24 hours. The reaction was stopped by repeated washing(10×12 ml) with DMF followed by methanol (3×12 ml) and ethanol (2×12ml). The resin was air dried prior to cleavage from the resin, asdescribed in the procedure above.

After the synthesis the peptides were cleaved from the resin using theReagent K as a cleavage mixture. Water (2.5%), TIS (2.5%) EDT (2.5%)were used as scavengers. The peptides were then precipitated with colddiethyl ether. The precipitates were centrifuged, washed three timeswith diethyl ether, dissolved in 20%-50% acetonitrile/water mixture andlyophilized. Analytical data of crude products were performed usinganalytical reverse phase HPLC and electrospray mass spectroscopy.

The crude peptides were purified by HPLC on a Vydac C18 or C4 column,2.5×25 cm, using a linear gradient of 10-50% acetonitrile in water, with0.06% trifluoroacetate (TFA) or with 0.1% triethanolamine acetate bufferpH7.8 (1%/min gradient, 10 ml/min flow rate). Monitoring by UV at 215 nmor 254 nm. Analytical HPLC was used to estimation or purity offractions. The final products were obtained as lyophilized with at least95% purity estimated by analytical HPLC (Vydac C18, 0.46×25 cm, lineargradient 10-60% acetonitrile in water, 0.1% TFA, 1%/min, 1 ml/min flowrate, detection by UV absorption at 215 nm and/or 254 nm. The purepeptides were identified by molecular mass analysis using a SCIEX APIIII mass spectrometer.

The fluorescence intensity of the labeled peptides were measured onBeacon Fluorescence Polarization System (Panavera) or Cytofluor 96 wellplate reader at gain 85.2 nmols sample of peptide dissolved in 50 ul ofDMSO, vortexing and aliquoted to final concentration of 1 uM with bufferEARLS (450 uL), pH 7.4. Intensity of fluorescence (Em. 450/50 nm-Ex530/25 nm) was measure for four concentrations in the range of 10-100nmolar. Linear regression was used for slope calculation. This value wasused to calculate of percentage of fluorescence of fluorescein itself asa sensitivity of specific response.

EXAMPLE 2

Pharmacological Binding and determination of IC50 for labeled peptides.

Samples were prepared for IC50 determination by dissolving 2-10 nmols ofthe sample into a final volume of 0.5 mL. In order to fully dissolve thepeptide, the sample was reconstituted in 20 uL DMSO, vortexing well toensure that all powder was completely dissolved, and then adding 0.48 mLof double distilled water. The stock solution was aliquoted to avoidrepeated freezing and thawing of peptide. Unused aliquots were stored at−20° C. (protected from light) for maximum 24 hr.

Receptor binding was carried out on tissues of rat submaxillary glandmembranes. Test were conducted in at least duplicate with three repeatsrecommended at 5-7 test concentrations (from 10⁻¹¹-10⁻⁶ M depending onthe binding values).

Data for native and labeled peptides were analyzed by non-linear curvefitting. This included statistical evaluation of fit and calculation ofIC50 and K_(i) values.

Other embodiments are within the scope of the following claims.

1 1 28 PRT Homo sapiens 1 His Ser Asp Ala Val Phe Thr Asp Asn Tyr ThrArg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser IleLeu Asn 20 25

What is claimed is:
 1. A compound of the formula:

wherein R₁ is a light-emitting moiety and R₂ is a VIP-based peptide,fragment, derivative or analog thereof, wherein R₂ is comprised ofHis-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-lle-Leu-Asn(SEQ ID NO: 1), said peptide being linked at an amino acid position to(C—X), wherein (C—X) is selected from the group consisting of C═O, C═S,CH(OH), C═C═O, C═NH, CH₂, CH(OR), CH(NR), CH(R), CR₃R₄, and C(OR₃)OR₄where R, R₃, and R₄ are alkyl moieties or substituted alkyl moieties,wherein said compound exhibits substantial biological activity in thepresence of a receptor having affinity for VIP-based peptides.
 2. Thecompound of claim 1 wherein said amino acid position is lysine.
 3. Thecompound of claim 1, wherein said amino acid position comprises a Lys15residue of said VIP-based peptide.
 4. The compound of claim 1 wherein R₁is bound, through C, to a region of said R₂ peptide which is notinvolved in said biological activity.
 5. The compound of claim 1 whereinsaid VIP-based peptide binds to a human receptor.
 6. The compound ofclaim 3 wherein said lysine residue of said VIP-based peptide isattached to (C—X) at an ε amine position.
 7. The compound of claim 1wherein said light-emitting moiety is selected from the group consistingof 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, fluorescein, FTC, Texasred, phycoerythrin, rhodamine, carboxytetra-methylrhodamine, DAPI,indopyras dyes, Cascade blue, coumarins, NBD, Lucifer Yellow, propidiumiodide, CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates,lanthanide chelates, non-fluorescent dialdehydes which react withprimary amines-to form fluorescent isoindoles, dansyl, fluorescamine anddabcyl chloride, 5-((((2-iodoacetyl) amino)ethyl)amino)naphthalene-1-sulfonic acid, long lifetime dyes comprised ofmetal-ligand complexes (MLC) and derivatives thereof.
 8. The compound ofclaim 1 wherein (C—X) is selected from the group consisting of C═O andC═S.
 9. The compound of claim 1 wherein said compound is apharmaceutically acceptable salt or complex thereof.
 10. A method forlabeling a receptor having an affinity for a VIP-based peptide bycontacting said receptor with the compound of claim
 1. 11. A method forgenerating a biologically active compound of claim 1, comprising:reacting R₁ and R₂ in an aqueous solution to form a mixture comprisingthe compound of claim 1 and secondary compounds having biologicalactivities less than 0.25% of the biological activity of R₂—H in thepresence of a receptor having affinity for VIP; contacting the mixturewith a receptor for VIP; and isolating from said mixture alight-emitting compound exhibiting substantial biological activity inthe presence of said VIP receptor.
 12. The method of claim 11 whereinsaid isolating step comprises: releasing said light-emitting compoundfrom said VIP receptor; and isolating said light-emitting compound. 13.The method of claim 12 wherein said step of isolating saidlight-emitting compound includes selection by high pressure liquidchromatography.
 14. A method for imaging cell receptor sites comprisingcontacting candidate cell receptor sites with a compound of claim 1, anddetecting said bound compound as an indication of said cell receptorsites.
 15. A method of cell sorting comprising contacting a populationof candidate cells with a compound of claim 1, and isolating cells boundto said compound.
 16. A method of flow cytometry comprising contacting apopulation of cells with a compound of claim 1 and detecting cellsbearing receptors on their surfaces by detecting cells bound to saidcompound.